The motor domain of the kinesin Kip2 promotes microtubule polymerization at microtubule tips

Chen et al. identify a function of the yeast kinesin Kip2’s motor domain in recruiting free tubulin and catalyzing microtubule assembly. They provide insight into the role of the yeast CLIP-170 in microtubule elongation, that is, to act as a cofactor to track microtubule plus-ends.

The microtubule plus-end directed molecular motor Kip2, in combination with the CLIP-170 homolog Bik1, has been shown to be important for cytoplasmic microtubule dynamics in budding yeast. The molecular mechanisms by which Kip2 promotes tubulin polymerization in cells and the contribution of Bik1 during this process, however, are not fully understood. In the current study, the authors use a combination of live cell imaging with high temporal resolution, in vitro biochemical and biophysical experiments and mathematical modelling to interrogate new aspects of the Kip2-Bik1 system. They show that the Kip2-NMD (non-motor domain) is sufficient for this interaction and that it depends on the c-terminal tail of the Kip2-NMD. Plus-end polymerization activity in vivo requires the motor domain of Kip2 and the authors detect binding to tubulin dimers in vitro. Structural homology models allow the authors to propose three positvely charged patches on the motor domain to be selectively involved in microtubule lattice binding, versus binding to tubulin dimers. Interestingly, mutation of patch 1 (P1), allows motility in vitro, plusend localization in vivo, yet seems to lack polymerization activity. The authors propose a model in which Kip2 promotes the incorporation by binding to free tubulin dimers, once it has reached the plus-end. Bik1 increases the retention of Kip2 at the plusend to support this effect. Overall this is an interesting study with a nice combination of in vivo and in vitro analysis. While there has been a fair amount of work on Kip2 already, there are a number of interesting novel aspects in this study.
Major points: 1. A novel aspect of the current study is the identification of the distinct patches P1,P2, P3 on the Kip1 MD involved in the binding of microtubules versus tubulin dimers. I missed the demonstration that these mutants indeed affect tubulin dimer binding in vitro (like in Figure 5A). This seems to me a crucial point in the current study, especially regarding the effect of the P1 mutation.
2. The use of an in vitro reconstitution assay for tubulin polymerization to demonstrate an effect of the Kip2 P1 mutation ( Figure  6) is appreciated. Along these lines it is a bit of a pity that the first part of of the study, which mapped crucial elements of the Kip2-Bik1 interaction, is not connected to these in vitro experiments in a similar manner. The authors cite that Bik1 is not required for Kip2 mediated polymerization in vitro, but there might be a quantitative effect of Bik1 in the reconstituted system? It seems that more features of the model proposed in Figure 7 could be tested in this setup.
Minor points:   I found the usage of the terms Kip2 NMD and Kip2 tail sometimes confusing. Sometimes NMD and Tail seemed synonomous for the non-motor part, sometimes "tail" only meant the extreme carboxyterminus. The authors should check again for consistency throughout the manuscript.
Page 5: Heading of the section: one should qualify the statement with "astral" microtubule growth in vivo Reviewer #2 (Comments to the Authors (Required)): In this manuscript, Chen et al. continue their investigation of the budding yeast kinesin polymerase Kip2, focusing on elucidating the molecular mechanisms underlying its microtubule polymerase activity. They employ a range of methods, from live-cell imaging of budding yeast, through biochemical and structural approaches to probe protein interactions, to TIRF-based reconstitution assays with purified protein components. The authors report that the c-terminal tail of Kip2 mediates interaction with its binding protein Bik1 (yeast Clip-170 ortholog), and that this tail domain is important for the Kip2's polymerase activity in vivo. They postulate that Bik1-Kip2 interaction contributes to Kip2's microtubule plus-end tracking and retention at plus ends in vivo. On the other hand, the authors report that Kip2's motor domain binds soluble tubulin, and that the residues involved in tubulin interaction (K294 & R296) are necessary for Kip2's polymerase activity in vitro. Taken together, the authors propose that Kip2-Bik1 interaction facilitates prolonged residency of Kip2 at the microtubule plus end, where motor domains of Kip2 facilitate microtubule polymerization through a direct interaction with soluble tubulin dimers.
The study is of high quality, and the results are presented in a clear and logical fashion. Several interesting questions and controversies remain. For example, in their previous work (Chen et al. 2019), the authors reported that Kip2 targeting to spindle pole bodies (SPB) depends on Bub2 and Bfa1, while Bik1 primarily targets microtubule plus ends. In this work, however, the authors conclude that Bik1 promotes Kip2 recruitment to SPBs (see Discussion), although it is not completely clear how that would work. Additionally, the conclusion that Bik1 increases Kip2's residency time at microtubule plus ends is not directly demonstrated, and the corresponding conclusions should thus be toned down. Finally, the authors are in a position to directly disprove a competing tubulin-shuttle model (suggested by Hibbel et al. 2015), which would provide significant support to the model proposed in this manuscript.
Specific comments: Figure 1: -It would be very insightful to show an example image/kymograph from which microtubule dynamics parameters (lifetime, growth and shrinkage rates) are extracted -Representation of microtubule growth and shrinkage rates on a log plot ( Figure 1G) is rather unintuitive -the authors should instead plot microtubule growth rates and microtubule shrinkage rates separately, both on a linear plot -Numbers of measurements should be added to the caption for all data (e.g. panels 1F and 1G)  : -This figure is the most confusing, while at the same time essential for the conclusions on how Kip2-Bik1 interaction encodes Kip2 localization in vivo. Since the intention is to directly compare wt vs delT Kip2 constructs in the presence and absence of Bik1, it would be more useful in 3B-D to plot intensity distributions for different mutants against each other on the same graph (e.g. just take 2.5 um long bin and plot the three distributions together) -The significance of slopes and intercepts in 3B-D is not discussed.
-While the authors have previously employed and extensively discussed their model (Chen et al. 2019), the significance of the modeling results in 3F-H is not clear and sufficiently elaborated. Modeling results should be overlaid with experimental data for direct comparison, as previously done in Chen et al. 2019. It is unclear to what extent the model predicts a unique set of effects on the modeled parameters. For example, could e.g. obtained differences in in-rate be compensated by differences in on-rate and/or concentration at SPBs? Is the 30% reduction in SPB body localization (established using ATP-deficient mutant) explicitly modeled? -Results presented in 3G should be plotted separately on a linear (not log) scale for clarity.
-Importantly, while 3H shows that modeling estimates a 2-fold increase in Kip2 out rate in the absence of Bik1 interaction, which corresponds to end-residency being reduced from 0.3 s to ~ 0.15 s, previous in vitro results (Hibbel et al. 2015) demonstrated that Kip2 residency times on plus ends on its own in vitro are two orders of magnitude higher (~30 s). Given such a large discrepancy between in vitro and in vivo results, it is hard to appreciate the potential effect that just a 2-fold modulation of the out rate might have. Figure 4: -The data in this figure (especially panel C) would be well supported by accompanying intensity linescans, allowing to directly compare investigated protein distributions/co-localizations.
-It is a little bit confusing that Kip2-NMD is targeted to plus ends via Bik1 interaction, but the full-length ATP-ase deficient Kip2 does not localize to plus ends in the presence of Bik1. Shouldn't this mutant still be able to bind Bik1 through its tail domain (have the authors checked this)? How is this observation explained?  Figure 6) at much lower Kip2-GFP and labeled tubulin concentrations (i.e. 100 pM-1 nM Kip2 and sub-micromolar tubulin range) which would directly reveal whether Kip2-tubulin interactions can occur along the microtubule lattice, and/or only at the ends).
-The evidence that Bik1 prolongs the residence time of Kip2 at ends is weak, as no direct measurements of residence times have been performed. This, of course, could be done using in vitro TIRF approach with purified Bik1. This may well be outside the scope of the manuscript, but in that case the authors should restrain from strong statements that this is indeed what is going on in cells (see Discussion).
Reviewer #3 (Comments to the Authors (Required)): The manuscript from Chen et al. addresses the important question of how kinesin proteins regulate microtubule (MT) dynamics overall, and specifically how the kinesin Kip2 promotes MT polymerization and stabilization. This activity is unclear because Kip2 requires its partner Bik1 to promote MT growth/stability in vivo, yet is capable of this alone in vitro. Bik1 itself is required for MT growth/stability in vivo. This manuscript investigates the mechanism of how Kip2 promotes MT growth and stability. The authors examine MT dynamics in cells and show that Kip2 likely promotes both MT polymerization and inhibits catastrophes. The various Kip2 truncations used are informative for the molecular regions required for MT growth, recruitment to SPBs, interaction with tubulin and with Bik1. The authors also test binding interactions between these regions and tubulin, MTs, and parts of Bik1 (CC domain). The authors find no evidence of binding of Kip2-NMT domain to tubulin or MTs using the construct from bacteria and conditions used. Interestingly, they do find that the CC domain of Bik1 does not interact with the CC of Kip2, but rather mainly via the tail region. Using a combination of in vivo imaging and mathematical modeling the authors present data that Bik1 likely recruits Kip2 to SBPs for transport along MTs, and prevents Kip2 from loading along the MT lattice. Bik1 also seems required for Kip2 remaining associated with MT +ends. Data also shows that the NMD of Kip2 recruits Kip2 to the SBP via Bik1, and that this effect may not occur directly at the +end, hence, Kip2 and Bik1 interactions originate the SPB. The authors identify three regions of positive charge on the MT-binding surface and mutate them, showing that P2 and P3 mutants fail to localize properly, whereas P1 mutants can. Although the P1 mutant localizes to +ends, the MTs are short as in a kip2 null. Thus, the P1 patch is needed to promote MT polymerization in vivo. The authors then test the function of Kip2-MD-P1 compared to WT Kip2-MD on MT polymerization and stability in vitro, with data suggesting that the P1 mutant is inefficient at promoting MT growth and stability. The mutant displays motility in vitro, so presumably this is because it lacks proper/robust interaction with free tubulin dimers. Although this could not be tested directly.
Overall this is a comprehensive body of work dissecting the MT polymerization and stabilizing activity of Kip2 in relation to Bik1 both in vivo and in vitro. It is well written, flows nicely, and data are high quality. The various mutants used are quite informative and the data support a model of Bik1 recruiting Kip2 to the SPB, Kip2 transporting Bik1 to the +end, then associating with the Bik1 pool at the plus end while using its motor domains to promote elongation of the +end. This is a significant conceptual advance regarding Kip2/Bik1 function. The data fall short of sufficiently supporting certain mechanistic aspects of the model.

Concerns:
1) A major mechanistic component of the model, that Kip2 motor domain binds curved tubulin to promote MT polymerization depends on the P1 mutant not binding to free tubulin. This should be tested at least in the SEC-MALS experiment as in Fig 5A. Moreover, the explanation on page 12 of why this interaction was not tested is unclear. Since this could not be done due to precipitation of the Kip2-MD-P1 mutant in tubulin buffer, and presumably a similar tubulin buffer was used for the TIRF and motility assays. Yet the SEC-MALS was in a Tris-based buffer.
2) The issue of precipitation also raises questions about the TIRF based experiments which support the direct involvement of P1 in MT polymerization. Equal molar concentrations of WT and P1 mutant were compared, with WT displaying more activity. P1 precipitation will result in lower apparent activity. How was the precipitation of the P1 mutant controlled for in the effective concentration? Is it known to what extent P1 was soluble under these conditions? 4) The authors use an automated system to measure MT length at ~1s intervals based on the distance between the centroid of Spc72 and Bik1 signals. It is unclear whether growth and depolymerization rates were calculated based on single timepoint changes in calculated length. More importantly, the criteria for how rescues and catastrophes were calculated based on these data is unclear. If they were calculated based on changes from positive to negative length changes within single timepoints, for instance, how can the authors control for potential changes in the shape and amount of Bik1 at the +end? Do the increased catastrophe events in kip2 delta cells lead to short-lived depolymerization events or more commonly to complete MT depolymerization? Fig S1B should be visualize using two colors as in Fig S1A. Particularly with the short spindle and aMT it is uncertain whether the two main spots represent 1 SPB and 1 +end or 2 SPBs. What is indicated as the second SPB is poorly visualized and also changing position. If this is due to 3D movements it clearly illustrates the limitations of single color imaging for multiple cellular structures. This result is critical for the interpretation of the Kip3-Kip2 chimera results in Fig 1D and the resulting conclusion that bringing the Kip2-NMD to the +end is not sufficient to restore MT growth without Kip2 motor domain.

5) The cells in
6) The data in Fig 4 A and C only show 1-2 cells per condition to back up conclusions in the text and needed to support subsequent findings. These results need to be quantified from a reasonable number of cells over multiple experiments. The images shown in Fig. 4C are also less than convincing that full length Kip2 is located at the MT +end in these cells.
7) The authors use labels at the 2 ends of the MT to infer length. Visualizing the MT may be slower but has the advantage of detecting MT bending, which can occur with longer MTs (e.g. in DeltaN cells). The authors should exclude the influence of MT bending from the conclusions and/or determine the frequency by also visualizing MTs in some cells.
Minor issues: 8) Why was the recruitment of Kip2-N-MD to SPB (or +end) by Bik1 not tested?
9) The authors should include some representative graphs of the MT length and lifetime measured with the method used in Fig  1. 10) In Fig 1 it is unclear if the graphs report maximum MT length of each MT, or the lengths of MTs accumulated from each time frame. The test indicates max length only but the figures and legend seem otherwise. Also, it should be clearly stated whether, if max length is graphed, how the authors eliminated multiple maximums following rescue events. We thank the reviewer for carefully reading our manuscript. We are very happy that the reviewer finds it interesting and appreciates the novel aspects revealed by our study.
Major points: Figure 5A). This seems to me a crucial point in the current study, especially regarding the effect of the P1 mutation.

A novel aspect of the current study is the identification of the distinct patches P1,P2, P3 on the Kip1 MD involved in the binding of microtubules versus tubulin dimers. I missed the demonstration that these mutants indeed affect tubulin dimer binding in vitro (like in
We have now succeeded in purifying the recombinant proteins MBP-Kip2-MD-mCherry and the corresponding mutant MBP-Kip2-MD-P1 --mCherry. Although the motor domain (MD) alone was difficult to work with, particularly when mutated, fusing it N-terminally to MBP and C-terminally to mCherry stabilized it and allowed us to work with it. SEC-MALS analysis of these proteins in the presence of unpolymerized tubulin are now shown in Figure 5CD. Beyond confirming that Kip2-MD binds unpolymerized tubulin, we now show that the P1 patch is required for this interaction. Figure 6) is appreciated. Along these lines it is a bit of a pity that the first part of of the study, which mapped crucial elements of the Kip2-Bik1 interaction, is not connected to these in vitro experiments in a similar manner. The authors cite that Bik1 is not required for Kip2 mediated polymerization in vitro, but there might be a quantitative effect of Bik1 in the reconstituted system? It seems that more features of the model proposed in Figure 7 could be tested in this setup.

The use of an in vitro reconstitution assay for tubulin polymerization to demonstrate an effect of the Kip2 P1 mutation (
We agree with the reviewer that it would be good to demonstrate the effect of Bik1 on Kip2mediated microtubule polymerization in vitro quantitatively. To address this point, we purified full length Kip2 fused to GFP and full length Bik1 fused to mCherry. In TIRF experiments with Bik1 and Kip2, we found that Bik1 binds microtubules poorly -less than half of the microtubules have bound Bik1. This is consistent with previous reports and could be explained in several manners. First, Bik1 binds C-terminal phenylalanine residues as in yeast tubulin but very poorly the Cterminal tyrosine of porcine tubulin used in our assay (see M. M. Stangier et al., 2018). This alone could already explain the difficulties of Bik1 binding microtubules in our in vitro assay. However, additional challenges affect this experiment further. CLIP proteins require EBs to track microtubule ends (K. A. Blake-Hodek, et al., 2010). The addition of Bim1 to the experiment and analysis of its own role would therefore be needed, which goes beyond the scope of this study. In any case, the addition of Bik1 in the tubulin polymerization assay did not affect microtubule dynamics, not surprisingly given these limitations. Given the inconclusive nature of these experiments, we have not added them to the manuscript.  Since the ITC data did not add substantial further insights to our main conclusion that "Bik1-CC interacts directly with Kip2-NMD and this stable interaction depends on Kip2's C-terminal tail" (page 7, line 4-5 of the revised manuscript), we have simplified the text and removed these data from the manuscript. The raw data are therefore no-longer relevant.

Figure 3: I found the order of the B and C to be confusing. Better to show the wildtype first?
We agree with the reviewer and are now showing wild type first.

Figure 5 A: I missed data for Kip2 MD alone and a corresponding analysis by SDS-PAGE of the SEC fractions.
We no longer have access to Kip2-MD proteins, which were very unstable, but have been able to produce MBP-Kip2-MD-mCherry. As mentioned above, we now show the data obtained with it in the new Figure 5C and Figure S1E.
I found the usage of the terms Kip2 NMD and Kip2 tail sometimes confusing. Sometimes NMD and Tail seemed synonomous for the non-motor part, sometimes "tail" only meant the extreme carboxyterminus. The authors should check again for consistency throughout the manuscript.
We now clearly define and only refer to the extreme C-terminus as the Kip2 tail.

Page 5: Heading of the section: one should qualify the statement with "astral" microtubule growth in vivo
We inserted 'astral' in the heading.

Reviewer #2 (Comments to the Authors (Required)):
In this manuscript, Chen et al. continue their investigation of the budding yeast kinesin polymerase Kip2, focusing on elucidating the molecular mechanisms underlying its microtubule polymerase activity. They employ a range of methods, from live-cell imaging of budding yeast, through biochemical and structural approaches to probe protein interactions, to TIRF-based reconstitution assays with purified protein components. We thank the reviewer very much for carefully reading and commenting on our manuscript. We are very pleased that the reviewer praises the quality of our study. We address below the specific concerns raised.
We have indeed previously demonstrated the function of Kip2 as a messenger deployed by yeast spindle pole bodies (SPBs) to control astral microtubule length and dynein distribution (Chen et al., 2019). In that study, we reported that Kip2 recruitment at the SPBs depends on Bub2 and Bfa1, but we did not investigate the contributions of Bik1. Therefore, that study did not exclude the possibility that Bik1 participates in any of the processes of Bik1 recruitment at SPBs and stabilization at microtubule tips. Indeed, Bik1 and Kip2 are both found at SPBs in addition to the plus-end of astral microtubules (Carvalho P. et al., 2004). It has been proposed that Kip2 meets Bik1 at the SPB and then transports it to microtubule plus-ends. But how Bik1 affects Kip2 localization and functions has been unclear, and this is one of the issues we address in this work. See our specific responses to the distinct points of the reviewer below. We thank the reviewer for this suggestion. We have inserted two new panels in Figure 1. Figure  1D shows an example of a preanaphase astral microtubule accompanied by kymographs and tracked trajectories. Figure 1E shows the corresponding plot of 3D aMT length as a function of time. We highlight how the maximum microtubule length, lifetime, speeds of growth, and shrinkage were extracted. We also added some examples of 3D astral microtubule length plotted as a function of time extracted from control and kip2∆ mutant cells in Figure S1AB.
-Representation of microtubule growth and shrinkage rates on a log plot ( Figure 1G) is rather unintuitive -the authors should instead plot microtubule growth rates and microtubule shrinkage rates separately, both on a linear plot.
As suggested, we plotted the speeds of the microtubule growth and shrinkage on a linear scale.
-Numbers of measurements should be added to the caption for all data (e.g. panels 1F and 1G) We updated the numbers of measurements on the panels.  Thank you for the suggestion, which we considered, but ultimately did not implement in the main text for the following reasons: -First, the distributions of Kip2 along aMTs differ not only between wt and mutants but also between aMTs of different lengths, such that picking only one length bin would neglect other important aspects of the data (please see also response below). -Second, we plotted these three graphs with consistent x-and y-scales to allow readers to compare across graphs. We now include an explicit statement when first referencing -The significance of slopes and intercepts in 3B-D is not discussed.
We now discuss the significance in the context of in vivo vs in vitro residence times at aMT plusends (see below). As suggested, we now provide an overlay of experimental data and simulation results (Supplementary Figure 3G to I). Comparisons of simulation and experimental data obtained for the WT and bik1Δ mutant cells indicate near-perfect matches, but peak densities for Kip2-∆T-3xsfGFP cells show deviations. Overall we conclude that the model adequately captures the data (see revised main text), and discuss the deviation in relation to the reviewer's other points on slopes/intercepts (above) and (Hibbel et al., 2015) (below).
Regarding unique sets of parameters, we have now added the following clarification: 'note that we used a Bayesian approach to estimate parameter values and their uncertainties; these uncertainties may be caused by measurements noise or by limited identifiability of individual parameters.' The justification for this statement is that, if the model were not identifiable, the kernel density estimates would indicate this. For example, k on and k in are harder to identify because the on / in rates depend on these parameters as well as the concentration of free Kip2 multiplicatively. As in our previous work (Chen et al, 2019), the key to identifiability is to fix Kip2's movement speed, which according to Fig. S2B is unchanged between wt and mutants at approximately 6.3 µm/min (consistent with (Chen et al, 2019)).
For the 30% reduction in SPB localization of Kip2, with the simplified model (the SPB is not modeled as an explicit compartment) and the available data (which cannot disentangle local Kip2 concentration at the SPB and transition rate constant to the microtubule), we do not see how to capture this effect in a principled manner. The current model captures only the total inrate and it is not clear how mutations influence the transition from the SPB. We now state this limitation explicitly.
-Results presented in 3G should be plotted separately on a linear (not log) scale for clarity.
We now provide a better explanation for this plot in log-scale and apologize for the omission.
We added the statement 'The modelling results first showed that in wild type and both mutants, Kip2's on-rate constants were orders of magnitude lower than its in-rate constants, previously observed (37). This implies that the primary recruitment of Kip2 from the microtubule minus-end (i.e., the SPB) is not fundamentally affected upon removing Bik1 or preventing Kip2 to interact with it. This interpretation is essential for the following discussion of differences between wt and mutants, and it requires a comparison of k in and k on estimates that is impossible in linear scale in one plot (together with differentiation between wt and mutants).
- To address the discrepancy of residence times with in vitro data, we first simulated the model with the k out value from (Hibbel et al., 2015), and all other parameter values unchanged. This resulted in predictions of 'traffic jams' at aMT plus-ends as shown in the simulated kymograph in the new Supplementary Figure 3J, which are clearly inconsistent with our in vivo data for Kip2.
For the related kinesin-8 Kip3, it is known that MT plus-end residence time decreases exponentially with (total) Kip3 concentration (1) and with mechanical force applied to these motors (2). Importantly, the in vitro experiment to quantify residence time in (Hibbel et al., 2015) used <1nM total Kip2, one to two orders of magnitude lower than our in vivo estimates (Fig. 3F). If Kip2 had a similar concentration and force dependence as Kip3, this would explain the difference between in vivo and in vitro residence times. It would also be consistent with (i) Decreasing peak densities (indicated by slopes in Fig. 3B-D) for (long) microtubules with high Kip2 density (indicated by intercepts in Fig. 3B-D), and (ii) mismatches between model and experimental data, particularly for long aMTs in Kip2-∆T-3xsfGFP cells where these uncaptured concentration effects would be most pronounced. We now provide this hypothesis and reasoning in the main text. We have generated and added line scan plots for panel C, now presented in panel D.

-It is a little bit confusing that Kip2-NMD is targeted to plus ends via Bik1 interaction, but the full-length ATP-ase deficient Kip2 does not localize to plus ends in the presence of Bik1. Shouldn't this mutant still be able to bind Bik1 through its tail domain (have the authors checked this)? How is this observation explained?
Indeed, the ATPase deficient Kip2 variant Kip2-G374A does not localize to plus-ends in the presence of Bik1. As shown in Figure 3C in Chen et al., 2019, in KIP2-G374A-3xsfGFP/KIP2-mCherry heterozygous diploid cells, Kip2-mCherry is enriched at the plus-end, and Kip2-G374A-3xsfGFP is absent from microtubule plus-ends. One of the main conclusions from that work is that Kip2 recruitment to microtubules in yeast cells is highly restricted to the SPBs (Chen. et al., 2019). Together, our previous and current data suggest that full length Kip2 cannot bind Bik1 at the microtubule plus-end (or the shaft for that matter) without first passing by the SPB. Now, the fact that Kip2-NMD can bind Bik1 at and localize to the plus-end of microtubules on its own comes indeed as a surprise. We suggest that some of the sequences present in the full length Kip2 and absent in the NMD construct restrict Kip2 from binding Bik1 at microtubule ends as long as it has not passed first by the minus end. For example, the disordered N-terminal domain of Kip2 (about 100 amino acids) and its phospho-regulation contribute to focusing the proteins recruitment to microtubules minus-ends (Chen et al., 2019). This domain is absent in the NMD construct. This might explain why the NMD and full length Kip2 show different prerequirements for them to bind Bik1 at microtubule plus-ends.

range) which would directly reveal whether Kip2-tubulin interactions can occur along the microtubule lattice, and/or only at the ends).
This is an interesting point. However, we are not ourselves convinced that our model and the shuttling model would be necessary mutually exclusive. The fact that the motor domain of Kip2 is able to directly bind and polymerize tubulin dimers at microtubule tips does not a priori exclude another part of the molecule from binding and transporting such dimers towards the microtubule tip. In any case, we have tried but not been able to visualize single molecules of tubulin dimers in TIRF experiments so far. Therefore, we cannot exclude that Kip2 would also transport tubulin dimers. ). This is a major technical obstacle that prevents us from performing this experiment (see also response to point 2 of reviewer 1). Therefore, we have not added these experiments to the paper. This being said, we do not think that the evidence that Bik1 prolongs the residence time of Kip2 at microtubule tips is that weak. Just looking at the images obtained in vivo clearly shows that the strong localization of Kip2 focused to the plus-end of astral microtubules in vivo disappears when Kip2 no-longer interacts with Bik1 (bik1∆ and Kip2-∆T mutant cells). Thus, Kip2 does stick for some time to the plus-end of microtubules and this requires Bik1 function. The simulations clearly support this notion and put numbers on it. Even if the effect might not seem huge, the point is strong: although the amount of Kip2 at microtubule tips is higher than in wild type due to an increased flow of Kip2 molecules towards the plus-end, in the bik1∆ mutant cells Kip2 leaves virtually as it arrives to the plus-end and this is sufficient for causing a consequent defect in microtubule polymerization. Thus, there is little doubt that Bik1 retains Kip2 at ends and that this is essential for Kip2 to function as a polymerase in vivo. We thank the reviewer for these very positive words. We agree with the points raised by the reviewer, which we addressed in full. Fig 5A. Moreover, the explanation on page 12 of why this interaction was not tested is unclear. Since this could not be done due to precipitation of the Kip2-MD-P1 mutant in tubulin buffer, and presumably a similar tubulin buffer was used for the TIRF and motility assays. Yet the SEC-MALS was in a Tris-based buffer.

Concerns: 1) A major mechanistic component of the model, that Kip2 motor domain binds curved tubulin to promote MT polymerization depends on the P1 mutant not binding to free tubulin. This should be tested at least in the SEC-MALS experiment as in
We now made new constructs, adding an MBP tag at the N-terminus and a mCherry tag at the C-terminus. These changes substantially increased the solubility and stability of the proteins, allowing us to successfully purify the recombinant proteins MBP-Kip2-MD-mCherry and MBP-Kip2-MD-P1 --mCherry. We performed SEC-MALS analysis of these proteins in the presence of free tubulin. The results are shown in Figure 5CD. We demonstrate that Kip2 MD indeed binds free tubulin and that the P1 mutation impairs Kip2-tubulin complex formation. The Kip2-MD proteins used in the SEC-MALS experiment shown in Figure 5A were not tagged and comprise only the motor domain alone. The TIRF experiments shown in Figure 6A-E were performed with MBP-Kip2 [1-560]-RFP. Not only these versions of the protein are fused to MBP and RFP, they are also longer than Kip2-MD [100-503]. This was refereed in pg.11 , now pg12): "At 2 nM concentration, purified wild type MBP-Kip2(1-560) fused to red fluorescent protein (RFP; Fig. 6AC; Table S2 and Fig. S1E, denoted Kip2-WT) strongly increased …". Under our experimental conditions, we did not observe protein precipitation with any of the recombinant proteins reported in the manuscript.

2) The issue of precipitation also raises questions about the TIRF based experiments which
3) Why does tubulin elute from the SEC column at 13.75 ml in Fig 2 and at 9.4 ml in Fig 5? Furthermore, why is Kip2-MD alone not analyzed in Fig 5? It is not possible to judge the strength of this interaction.
We used a Superdex200 SEC column for all but the experiment shown in Figure 5A, which was performed with a Superdex75 SEC column. The method section has been completed accordingly. For what concerns MBP-Kip2-MD-mCherry, we now show the analysis of the protein alone in Figure 5C.

4) The authors use an automated system to measure MT length at ~1s intervals based on the distance between the centroid of Spc72 and Bik1 signals. It is unclear whether growth and depolymerization rates were calculated based on single timepoint changes in calculated length. More importantly, the criteria for how rescues and catastrophes were calculated based on these data is unclear. If they were calculated based on changes from positive to negative length changes within single timepoints, for instance, how can the authors control for potential changes in the shape and amount of Bik1 at the +end? Do the increased catastrophe events in kip2 delta cells lead to short-lived depolymerization events or more commonly to complete MT depolymerization?
We use a semi-automated system to record and document the dynamics of microtubules in living cells. The catastrophe and rescue events were annotated manually based on the global microtubule length profile rather than length changes between two time points (1.07 to 2.14 s). We now present representative examples of 3D astral microtubule length plotted as a function of time extracted from control and kip2del cells in Figure S1AB. We also state that the catastrophe and rescue events were manually annotated. Increased catastrophe events in kip2∆ cells more commonly lead to complete microtubule depolymerization.
We also inserted two new panels in Figure 1. Figure 1D shows an example of a pre-anaphase astral microtubule accompanied by kymographs and tracked trajectories. Figure 1E shows the corresponding plot of 3D aMT length as a function of time. We highlight how the maximum microtubule length, lifetime, speeds of growth, and shrinkage were extracted. Fig 1D and the resulting conclusion that bringing the Kip2-NMD to the +end is not sufficient to restore MT growth without Kip2 motor domain.

5) The cells in Fig S1B should be visualized using two colors as in Fig S1A. Particularly with the short spindle and aMT it is uncertain whether the two main spots represent 1 SPB and 1 +end or 2 SPBs. What is indicated as the second SPB is poorly visualized and also changing position. If this is due to 3D movements it clearly illustrates the limitations of single color imaging for multiple cellular structures. This result is critical for the interpretation of the Kip3-Kip2 chimera results in
All our time-lapses were recorded in single color to achieve high temporal resolution. Very few haploid Kip3-Kip2 chimera cells made visible aMTs. For the cell shown in Fig S1B, now Fig S1D, the microtubule plus-end started above the bud-neck at the first time point. Shrinkage of the microtubule then brought the plus-end across the bud neck down to the mother cell. In contrast, the SPBs were less mobile and never crossed the bud neck into the bud. To strengthen our evidence, we now include a representative movie (movie S2) of the cell shown in Fig S1D. In the movie, the microtubule plus-end and the spindle poles can be differentiated with excellent confidence.

6) The data in Fig 4 A and C only show 1-2 cells per condition to back up conclusions in the text
and needed to support subsequent findings. These results need to be quantified from a reasonable number of cells over multiple experiments. The images shown in Fig. 4C are also less than convincing that full length Kip2 is located at the MT +end in these cells.
These results illustrated in figure 4A are now quantified for substantial cohorts of cells and reported in the new Figure 4B.
Images for Kip2-G374A-3xsfGFP/Kip2-mCherry diploid cells in the previous Figure 4C (now Figure 4D) were challenging to acquire since mCherry is not as bright as 3xsfGFP. We increased the exposure time for the mCherry channel from 30 ms to 50 ms. We now provide the line scan analysis of the microtubules to demonstrate the relative localization of Kip2-G374A-3xsfGFP and Kip2-mCherry.

7) The authors use labels at the 2 ends of the MT to infer length. Visualizing the MT may be slower but has the advantage of detecting MT bending, which can occur with longer MTs (e.g. in DeltaN cells). The authors should exclude the influence of MT bending from the conclusions and/or determine the frequency by also visualizing MTs in some cells.
This is indeed an important point. We have also visualized these microtubules using tubulin labels. Among all mutants reported in this work, aMT bending was only observed in Kip2-∆N pre-anaphase cells. We excluded those cells from our analysis and clarified this point in the methods section. Since including those cells will only further increase the length of aMTs in Kip2-∆N pre-anaphase cells, it would not change our conclusions.
Minor issues:

8) Why was the recruitment of Kip2-N-MD to SPB (or +end) by Bik1 not tested?
We did not test this mutant in living cells as Kip2-N-MD would not dimerize. We did express Kip2-N-3xsfGFP from the endogenous KIP2 locus, the resulting proteins were diffusive and did not localize to any specific structure. Fig 1. We followed this excellent idea and inserted panel DE to Figure 1 to illustrate how the maximum aMT length, lifetime, and speeds of growth and shrinkage were extracted. We further present more examples of 3D aMT length as a function of time in Figure S1. Fig 1 it is  Indeed, we report maximum aMT length in our tables and graphs. For each aMT, we generate only one value of maximum length, regardless of the number of rescue events. We have now clarified this in our captions and methods. Fig. 3G. The graphs are somewhat busy, and so it should be specifically noted in the text as for the statistical significance of Kip2-GFP in bik1delta cells, the results for the deltaT cells, while similar effects, changes in neither parameter are statistically significant. The present wording could be somewhat misleading.

11)
Thank you for spotting this imprecision -we have revised the statement.
12) The data/model presented in this manuscript would be strengthened by context with the recently demonstrated remote control mechanism regulating Kip2 loading and transport on MTs (Chen et al., 2019, eLife). In this manuscript it was shown that phosphorylation of Kip2 N region restricted Kip2 loading to SPBs. Is this mechanism independent of those shown in the current manuscript, upstream, or perhaps synergistic?
In (Chen et al., 2019, eLife), we reported that phosphorylation of Kip2's N-terminus prevents Kip2 from landing along microtubule lattices by ablating phosphorylation with a S63A point mutation (Kip2-S63A). In the current study, we removed the N-terminus to generate the mutant Kip2-∆N. Both mutants presented longer and longer-lived astral microtubules, which suggests that Kip2's N-terminus negatively regulates Kip2's microtubule polymerization activity. Since the S63A mutant shows a similar phenotype, our data suggest phosphorylation represses the inhibitory role of the N-terminus. Interestingly, the inhibitory activity of Kip2's N-terminus is nolonger observed in the absence of Kip2's C-terminal tail ( Figure 1CF). Fusing the N-terminus upstream of the NMD fragment dampened the ability of Kip2-NMD to target microtubule plusends and SPBs (unpublished result). This effect was reduced when the serine S63 was mutated to alanine. We can conclude that Kip2's N-terminus is a negative regulator of the microtubule polymerase activity of Kip2 and of Kip2 accumulation at microtubule tips. What the mechanism of this inhibition is and how it relates to the phosphorylation-based restriction in Kip2 loading onto microtubule shaft are open questions that will require extensive work and extend beyond the scope of the present study. Thank you for submitting your revised manuscript entitled "The motor domain of the kinesin Kip2 promotes microtubule polymerization at microtubule tips." We would be happy to publish your paper in JCB pending final revisions necessary to meet our formatting guidelines (see details below) as well as the text changes requested by Reviewer #3 to more clearly differentiate between conclusions based on data and those based on modeling simulations.
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1 Zürich, March 10, 2023 Dear Editors, It is with great happiness that we have learned about your decision to publish our manuscript "The motor domain of the kinesin Kip2 promotes microtubule polymerization at microtubule tips" in the Journal of Cell Biology. We have now gone through your final requests and edited the text and figures accordingly. A response in which we explain how we adapted the text to satisfy the last concerns of reviewer #3 can be found below. We thank you very much for publishing our paper, which we are very much looking forwards to see in print.
With my best regards,

Yves Barral
Response to reviewer #3: In general, the authors have done a nice job of addressing the reviewer concerns. I have a few concerns remaining that I feel can be addressed with text revisions, but also feel it is important they are addressed prior to publication. Several of my comments are in the vein of one of Reviewer 2's prior concern, in that statements about binding rates and dwell times of Kip2, which were inferred from varying parameters in simulations are conflated with actual observations and stated/discussed without proper qualification.
Regarding the general role of modeling in this work, we wish to clarify (see below for details) that we used the model primarily as an analytic tool for estimating parameter values from in vivo observations. This function of our model is identical to the case where we use observations such as spatial positions of microtubule tips at discrete time points and a linear regression model to estimate parameters that are not directly observable, such as microtubule growth and shrinkage rates. For our model based on ordinary differential equations, this estimation involves simulations because a closed-form solution as in linear regression is not possible, and by multiple simulations in a Bayesian approach, we obtain both the parameter estimates and their uncertainties. Our model itself was previously validated (ref) with in vivo data and it is based on physical constraints (mass balances), chemical kinetics, and measured parameters (e.g., motor speeds) whenever possible, making it suitable for the inference we are performing here. Finally, we only used the model for prediction instead of inference in one case, when evaluating the predicted impact of in vitro parameters on Kip2 distributions (bottom of page 8).
1) The modeling results in the results and discussion sections are presented as definitively disclosing what is occurring in vivo. This aspect is significantly overstated. At best, the modeling results are consistent with some possible scenario, or suggestive of what might be occurring in vivo. These instances of modeling results must be framed in the proper context. As an example, the top of page 9 states: "In addition to this, the modelling results showed that in wild type cells and both mutants, Kip2 is much more likely to start a run along the microtubule at its minus end than at any other position on the microtubule: Kip2's on-rate constants were orders of magnitude lower than its in-rate constants, as previously observed (37). Thus, disrupting the Bik1-Kip2 interaction did not affect dramatically Kip2 recruitment at the microtubule minus-end, i.e., the SPB. The estimates, however, indicated as well that Kip2's on-rate and in-rate constants were both significantly increased in bik1Δ and to a lesser extent in the Kip2-∆T-3xsfGFP mutant cells (Fig. 3G)...". The modelling did not show what Kip2 does in cells. It can show what Kip2 'may' do in cells, and real experimental evidence is needed to support the possibility. This is not made clear in sections of the results and discussion.
We have revised the text (p.8, 1 st para) to specify the role of the model with the rationale above. To distinguish inferred results from direct observations, the text now uses 'inferred parameter values' (i.e. values estimated using the dynamic model and in vivo observations) consistently. For example, the sentence quoted by the reviewer now reads: "In addition to this, our estimates for Kip2's on-rate constants were orders of magnitude lower than its inrate constants, as previously observed (37). Thus, in wild type cells and both mutants, Kip2 is much more likely to start a run along the microtubule at its minus-end than at any other position on the microtubule." Regarding 'real experimental evidence', we refer to the rationale above -parameters such as on-rate constants are not directly observable, neither in vivo nor in vitro.
2) Page 14 the discussion states: "The second is a binding site for the cytoplasmic linker protein Bik1 that increases Kip2 residence time at microtubule plus-ends in living cells, in a Bik1-dependent manner." It should be clear what aspects of this conclusion/discussion are supported by results obtained in vivo/vitro, and which are inferred from varying simulation parameters.
We have re-phrased the sentence to: "The second is a binding site for the cytoplasmic linker protein Bik1 that, according to our estimates, seems to increase Kip2 residence time at microtubule plus-ends in living cells, in a Bik1-dependent manner." With the consistent use of "estimates", we hope that the context is now clear.
3) Similarly in the second paragraph on page 15 statements that are based on simulations are convoluted with statements based on observations in vivo and in vitro. The basis for such statements should be clear when they are based on output generated by simulated scenarios. For example: "Both the loss of this interaction or Bik1 altogether decreases the time Kip2 spends at microtubule plus-ends." The basis for this statement was given in the next sentence (now combined as one sentence to clarify this basis): " … our out-rate constant estimates for Kip2 dissociation are consistent with Kip2 dissociating as soon as it arrives at the microtubule plus-end when Bik1 is absent." 4) Another example from the middle of page 15: "Thus, for Kip2 to promote microtubule growth, it needs to linger around the microtubule plus-end for some time, in a Bik1dependent manner." This statement as a conclusion may be OK, as long as the previous inferences that are based on simulations with altered parameters are clearly noted as described above. Otherwise, a statement like this should be qualified such as "We propose" or "Our modeling results indicate Kip2 likely needs to linger..."